Omega-7 fatty acids are monounsaturated fatty acids with a double bond residing at the seventh carbon atom. Palmitoleic acid (C16:1) is one of the omega-7 fatty acids, which is represented by the following chemical formula: CH3(CH2)5CH═CH(CH2)7COOH. Palmitoleic acid is biosynthesized from palmitic acid by the catalytic action of delta-9 desaturase.
Palmitoleic acid has a significant application value in the fields of medicine, nutrition, industry, etc. For example, palmitoleic acid has been proved to be able to increase the human body's sensitivity to insulin and be effective for diabetes and metabolic syndrome without obvious side effects (Cao H M, et al. Identification of a lipokine, a lipid hormone linking adipose tissue to systemic metabolism. Cell, 2008, 134, 933-944).
Not only that, palmitoleic acid is also able to reduce the level of C-Reactive Protein (CRP), and lower the risks of heart diseases and stroke by reducing inflammations. Moreover, palmitoleic acid may increase the fluidity of the cell membrane, decrease the content of low-density lipoprotein (LDL) cholesterol in blood, and reduce vascular occlusions resulting from the formation of atherosclerotic plaques in blood vessels, thereby preventing arrhythmia and reducing high blood pressure, etc. (Akazawa Y, et al. Palmitoleate attenuates palmitate-induced Bim and PUMA up-regulation and hepatocyte lipoapoptosis. J Hepatol, 2010, 52, 586-593. Misra A, et al. Obesity, the metabolic syndrome, and type 2 diabetes in developing countries: role of dietary fats and oils. J Am Coll Nutr, 2010, 29, S289-S301).
Furthermore, palmitoleic acid has excellent percutaneous permeability, therefore, it also has obvious effects in preventing skin aging and fat deposit, restoring the skin's elasticity, etc., and is an ideal raw material for anti-aging cosmetics.
In addition, palmitoleic acid may be directly used in high-efficiency production of greatly demanded industrial octylene (Rybak A, et al. Acycli diene metathesis with a monomer from renewable resources: control of molecular weight and one-step preparation of block copolymers. Chem Sus Chem, 2008, 1, 542-547). Besides, since palmitoleic acid is a monounsaturated fatty acid and has outstanding characteristics of low-temperature resistance and oxidation resistance, it is fit for producing high-quality biodiesel (Cao Y J, et al. Production of free monounsaturated fatty acids by metabolically engineered Escherichia coli. Biotechnolgy for Biofuels, 2014, 7, 59. Knothe G, et al. Biodiesel derived from a model oil enriched in palmitoleic acid, Macadamia nut oil. Energy Fuel, 2010, 24(3), 2098-2103).
So far, the main raw materials of commercially available palmitoleic acid products at present are wild plants. Many companies choose to extract and separate palmitoleic acid from Hippophae rhamnoides. 25% palmitoleic acid may be accumulated in the pulp of the Hippophae rhamnoides (Yang B, et al. Fatty acid composition of lipids in sea buckthorn (Hippophae rhamnoides L.) berries of different origins. J Agric Food Chem, 2001, 49, 1939-1947). Doxantha unguis-cali and Macadamia integrifolia are also important sources of palmitoleic acid. The content of palmitoleic acid in the seeds of Doxantha unguis-cati is about 60%, and that in the Macadamia integrifolia is about 30%. In addition, mink oil also contains little palmitoleic acid.
However, these wild plants and animals rich in omega-7 fatty acid cannot be planted and commercially produced in large scales like common oil crops due to such reasons as limited resource amount, low yield, narrow geographical distribution or rare population. Main components of seeds of oil crops such as soybean and corn planted in large scales at present include palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1) and linoleic acid (C18:2), and these seeds contain merely trace amount of (less than 2%) palmitoleic acid (Cao Y J, et al. Production of free monounsaturated fatty acids by metabolically engineered Escherichia coli. Biotechnolgy for Biofuels, 2014, 7, 59. Imke L, et al. Fatty acid profiles and their distribution patterns in microalgae: a comprehensive analysis of more than 2000 strains from the SAG culture collection. BMC Plant Biology, 2011, 11, 124), which may not meet the requirements of human beings in eating and industry (Table 1). In recent years, it has been found that fatty acids of some yeasts (e.g., Kluyveromyces polysporus, Torulaspora delbrueckii, and Saccharomyces cerevisiae) include palmitoleic acid in a high ratio. Nevertheless, these yeasts are low in total lipid content, leading to a low ratio of the content of palmitoleic acid in dry cell weight (Beopoulos A, et al. An overview of lipid metabolism in yeasts and its impact on biotechnological processes. Applied Microbiology and Biotechnology, 2011, 90, 1193-1206. Liu Y, et al. Bioconversion of crude glycerol to glycolipids in Ustilago maydis. Bioresource Technology, 2011, 102, 3927-3933).
TABLE 1Composition of Fatty Acids of Several Typical Oil CropsOil14:016:016:118:018:118:218:3SoybeanNR 11%NR4.0%23.4%53.2%7.8%CornNR10.3%NR1.0%30.3%58.2%NRRapeseedNR 5.5%NR2.2%58.3%19.9%9.1%JatrophaNR15.3%1.3%4.1%38.4%43.4%NRPalm1.0%42.8%NR4.5%40.5%10.1%0.2%
As the advantages of the palmitoleic acid have been increasingly discovered and proved, the demand for the palmitoleic acid will increase significantly. Therefore, seeking for a new resource high in palmitoleic acid content and unrestricted by seasonal and regional factors becomes the first choice to solve the problem of insufficient supply of raw materials.
Tribonema sp. belongs to Tribonema, Tribonemataceae in Heterotrichales under Xanthophyceae in Xanthophyta, and its frond is non-branched filament. Researches show that Tribonema sp. has the characteristics of high growth speed and high lipid content, and the content of palmitoleic acid in its fatty acids profile exceeds 50% (Wang H, et al. Integration process of biodiesel production from filamentous oleaginous microalgae Tribonema minus. Bioresour Technol, 2013, 142, 39-44. Guo F J, et al. Special biochemical responses to nitrogen deprivation of filamentous oleaginous microalgae Tribonema sp. Bioresour. Technol. 2014, 158, 19-24.). Therefore, Tribonema sp. may become a new raw material for producing the palmitoleic acid instead of the traditional wild plants and animals. Meanwhile, it is imperative to develop a method capable of cultivating Tribonema sp. in high density and in large scales.
There are two major ways for large-scale cultivation of microalgae: autotrophy and heterotrophy, wherein by the way of autotrophy, greenhouse gas carbon dioxide can be immobilized and oxygen is released; it is environmentally friendly; however, due to shading effect between the microalgae cells, the utilization of light energy is often greatly restricted. The higher the cell concentration is, the more obvious the shading effect is, which leads to a strong influence on the growth of cells and lipid synthesis. Concerning heterotrophy, the growth of cells relies largely on cells absorbing organic carbon sources; as it is not restricted by light, quick increase of the cell density and high-efficiency lipid synthesis can be achieved by adding organic carbon; however, the protein and pigment contents in heterotrophic algal cells are low when compared with photoautotrophy. Therefore, the above-mentioned two large-scale cultivation modes of microalgae have respective advantages and disadvantages, and should be selected according to specific requirements in practical application.
American patent No. US2013/0129775A1 discloses a composition rich in omega-7 fatty acid and a method of separating omega-7 fatty acid. While this patent sets forth that the composition is derived from algal biomass, the algal type is not specified, and brief description is only made with Nannochloropsis as an example.
American patent No. US2014/0275596A1 discloses a blend composition of algal omega-7 and omega-3 fatty acid. While this patent lists multiple algae as sources for producing omega-7 and omega-3 fatty acid blend compositions in detail in its detailed description of the invention, Tribonema sp. is not included.
Documents “Application of sweet sorghum for biodiesel production by heterotrophic microalga Chlorella protothecoides” (Gao C F, et al. Application of sweet sorghum for biodiesel production by heterotrophic microalga Chlorella protothecoides. Applied Energy, 2010, 87, 756-761.) and “Enhancement of microalgal biomass and lipid productivities by a model of photoautotrophic culture with heterotrophic cell as seed” (Han F F, et al. Enhancement of microalgal biomass and lipid productivities by a model of photoautotrophic culture with heterotrophic cell as seed. Bioresour. Technol, 2012, 118, 431-437) report a method of cultivating microalgae by heterotrophic culture, but the microalgae cultivated by heterotrophic culture is unicellular Chlorella. Those skilled in the art should know that the collection process of the Chlorella will consume high electric energy or require the addition of a certain quantity of flocculant. Tribonema sp., in the filamentous form, can be collected efficiently through simple filtration by bolting silk or air floatation; thus, the cost of collection is reduced, and the addition of flocculant may also be effectively avoided.
Concerning large-scale cultivation of Tribonema sp., as the existing researches are all based on photoautotrophy with no research made on heterotrophy or mixotrophy thereof, and the researchers in this art alway focus on heterotrophy of unicellular algae and seldom on multicellular filamentous algae, Tribonema sp. is cultivated and industrially used in the mode of autotrophy at present. For example, Chinese patent No. CN103960117A discloses a method for preparing Tribonema biological oil, and the Tribonema biological oil prepared through the method. In the method disclosed by this patent, a commonly used microalgae culture medium (i.e., BG11 medium) is employed to cultivate Tribonema under light to obtain Tribonema biomass; the growth pattern of the Tribonema sp. is photoautotrophic growth and the Tribonema biological oil is also derived from Tribonema biomass obtained through photoautotrophic growth; however, restricted by such factors as environment, season, and capacity, and the shading effect between cells, the growth rate of the Tribonema sp., the reachable cell density, yield of biomass and yield of lipid of the Tribonema need to be further increased.